Wednesday, May 28, 2014

Dissection Guides

   
Starfish
The external anatomy: 
The first thing we did was determine which side was the top and which was the bottom. The side that was more rounded and had the madreporite close to the middle was the top, and the side with an indent for the mouth was the bottom. Then, we found the axis of symmetry of the starfish. All of these features are labelled below. Other features on the exterior of the starfish are also labelled. The spines can be seen on both the top and bottom, and are used for protection. Our starfish had five arms, each with an ambulacral groove containing tube feet. These allow the starfish to move. Then, there is the central plate. This is at the center of the top side, and contains the madreporite. This is a bony spot that is also used for protection.

The internal anatomy: 
On the inside, the features that line each arm include the digestive tracks, the ambulacral ridge, and ampullae. The digestive tracts allow the starfish to process nutrients that are received from the stomach. The stomach is in the middle of the starfish, below the skin and bony madreporite. The ambulacral ridges, which link to the ring canal, transport water throughout the body of the starfish. There are also gonads at the top of the ampullae, which line the ambulacral ridges. These are like the sex organs of the starfish. 

Incision guide for dissection: 
The first step to dissect a starfish is to determine the line of symmetry. Then, cut off the end of one of the arms. Now, you can see the skin and the end of a digestive tract. Make a rectangle-shaped cutout that only pierces the skin, and you can see the top of the digestive tract. If you pin back the skin, you can see the top of the digestive tract. Upon removing the digestive tracts, you can see the ambulacral groove lined by ampullae. If you follow the groove towards the center, you can see the small gonads. The next step is to cut around the madreporite to create a circular flap of skin. When this is removed, you can see the stomach. The mouth is under the stomach.

Dissection Video:
http://m.youtube.com/watch?v=VBNOCCL4l5s

Clam
The external anatomy:
The first thing we had to do was to find the anterior and posterior sides of the clam. Next, we located the umbo, as well as the dorsal, lateral and ventral surfaces. The lines on the outside of the shell are called growth rings, and they tell us how long the clam has been alive. 

The internal anatomy:
Once the clam is opened, we can find the anterior and posterior adductor muscles. These muscles are responsible for opening and closing the clam, much like the jaw muscles of humans. Next, we find the gill. This is what the clam uses to breathe. Next to the gill is the foot, which is used to burrow into mud or sand. Under the gill and the foot we find the yellowish, spongy, reproductive organs. 

Incission guide for dissection:
The first step of dissecting a clam is to determine the anterior, posterior, ventral, and dorsal sides of the clam. Place the clam on its dorsal side and insert a screwdriver into the clam. Carefully work the screwdriver back and forth to losen the jaws. Pry the clam open and look at the internal organs. Locate the anterior and posterior adductor muscles and the gill and the foot and reproductive organs. 








Dissection Video:
http://m.youtube.com/watch?v=qfMVg3Zrrr8


Frog

The External Anatomy:
To decide whether the frog we had was a male or female we looked at the frogs hands to see if it had enlarged thumb pads or not. Our frog did, meaning that it was a male. The frogs external features on its head are its external nares, which the frog uses when it is immersed in water. Frogs normally just breathe out of their mouth, but the nares allow it to swim with their external breathing points only outside of the water. The mouth is what the frog uses for most of its breathing and eating. The tympani are the frogs structure for hearing. The eyes of a frog do not move like human eyes, their eyes bulge out from their head so much so they can see in different directions at once. The nictitating membranes are basically a third eyelid for the frog that is used for extra protection when the frog is outside of the water.
The Internal Anatomy:
Frogs have two types of teeth: vomerine and maxillary. The vomerine teeth are mostly vestigial in frogs. Their function is to hold and capture prey. The maxillary teeth are small and sharp and located near the upper jaw. Their functiom is to chew and crush prey. There maxillary teeth are not really used that often because in most cases frogs swallow their prey hole and don't chew them. Internal nares are a frogs nostrils that are used when their mouths are closed to breathe. Their tong mostly aids in grabbing their prey. The Eustachian tubes balance pressure in the frogs inner ear while it is swimming. The glottis is the tube leading to the frogs lungs and the esophagus is the tube leading to the stomach. The pharynx  is what food, liquid, and air passes through. We were not able to get a good picture of the mouth. But we were able to get one picture with the tongue and the maxillary teeth. 
The organs of the digestive system of the frog are the cloaca which is found between the hind legs of the frog and sperm, urine, and feces exit out of and then the esophagus, stomach, small intestine, large intestine, liver, gallbladder, and pancreas. The heart is located above all of these organs. 

Incision Guide for Dissection:
The incision we made for the frog was at the opening of the cloaca, between its hind legs. Then we cut all the way up to the frogs mouth on the underside and then at each leg we made a cut as well so we could pull back the skin to pin it down and get a better view. We used scissors instead of a scalpel so we wouldn't pierce any of the internal organs. We cut through the muscles and breastbone to be able to look at the internal organs. 
  

      Dissection Video:

http://m.youtube.com/watch?v=8KH6e26sPhI

Perch 
The external anatomy:
The three main sections of the fish are the head, trunk, and tail. The pectoral, dorsal, pelvic, anal, and caudal fins cans all be located on the exterior. The lateral line can also be located on the side of the perch. Next to the eyes the gill chamber can be found, covered by the bony operculum.  The nostrils are in front of the eyes. If you open the mouth, the teeth of the perch can be seen. The fish itself is covered in scales; the scales, if seen under a microscope, have lines that indicate age. 

The internal anatomy:
The cream covered liver is at the front of the body cavity. The gall bladder is between the lobes of the liver. Under the gall bladder and liver, the asophagus can be seen attaching to the stomach. At the posterior end of the stomach are the coiled intestines. The spleen is the small reddish-brown organ near the stomach. Below the operculum are the bony gill rakers. In front of the liver and below the gill rakers is the heart. The heart has two chambers: the atrium and the ventricle. Below the lateral line is the swim bladder, which gives the fish buoyancy. Below the swim bladder are the gonads, and testes/ovaries. The kidneys are the two long, dark organs near the posterior end of the perch. These filter its blood. 

Dissection guide:
Take pins to secure the fish. Use scissors to cut off the operculum, so that the gill rakers can be seen. One can be removed to observe. Now, make a rectangle cut under the lateral line. It should extend close to the head of the fish, as this is where most of the perch's organs are. Cut through both the skin and muscle, removing scales if need be. Removing the flap of skin makes these organs visible. 




Dissection Video: 


http://m.youtube.com/watch?v=tGSz1UTeQ90

Thursday, March 6, 2014

PGLO Lab

Purpose
Thre purpose of this lab was to find the transformation efficiency of the plasmid in E. Coli in different mediums. The independent variables were the presence of plasmid and the type of medium. The different mediums were +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, and -pGLO LB. The dependent variable was the transformation efficiency. 

Introduction
Transformation efficiency is the total number of cells growing on the agar plate divided by the amount of DNA spread on the agar plate, and it shows approximately how many cells take on the the DNA of the plasmid. Plasmid was the DNA that was inserted into the E. Coli, and it was called pGLO. Heat shock is the procedure used to form holes in the plasma membrane of the E. Coli. 

Methods: First off, we labeled two micro test tubes with +pGLO and -pGLO. Then with a new pipet, we put 250 micro-liters of the transformation solutions which is CaCL 2 into both of the test tubes. Then after adding the transformation solution we had to put the tubes in a cup of ice.  We had to then go over to the starter plate and get a single colony of bacteria and put it into the +pGLO tube.  We had to spin the loop with the bacteria on it in the tube to get it off and make sure all of it stayed in the tube. Then, we repeated the same thing for -pGLO. Only for the +pGLO tube we put pGLO plasmid DNA into it. Then, we put the tubes back in the foam rack and put it in an incubator for 10 minutes. While we were waiting we labeled four LB agar jars:  +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, -pGLO LB. we put the tubes into a heart shock for 50 seconds, which was set at 42 degrees Celsius, then we had to put them on ice for 2 minutes. We took the tubes off the ice and with a sterile pipet, added 250 micro-liters  LB nutrient broth into both the the tubes and let them sit at room temperature for 10 minutes. Once again with a new pipet, we put 100 micro-liters  transformation and control suspensions onto the agar plates. Using a new sterile pipet for each plate, we spread the suspensions evenly around the surface. Then we stacked our plates and put them in a 37 degree celcius incubator overnight.  

Data
We took our data from the pGLO positive LB/Amp/Ara plate in order to determine the Transformation efficiency. This would tell us how many cells out of our total maintained the plasmid.
Using all of this data, we determined that there are 2.17*10^3 transformants per microgram. This means that approximately 2170 cells in each microgram take on the plasmid and its traits. The ones that take on the plasmid will have the ampicillin resistance and glow in the dark trait. 

Discussion
This lab was only possible because of the heat shock. When a heat shock occurs, it allows cellular membranes to pull apart just enough to let in a plasmid. This is how the pGLO plasmid entered into the E. Coli cells, allowing the traits to be adapted. When pGLO was not present, however, growth depended on the presence of ampicillin. With ampicillin in the growth medium, there was no growth of E. Coli cells. Without ampicillin, there is one large colony of E. Coli growing unimpeded.  But when pGLO is incorporated into the cell, E. Coli gains the trait of ampicillin resistance. The plasmid also allows the bacteria to glow in the dark, but only when supplied with sugar. This is why one plate, the one without sugar, has normal E. Coli colonies while the other, with sugar, was able to glow. The last two plates, with both pGLO and ampicillin, have sparse colonies compared to the unimpeded growth of the first. This is because the plasmid is not incorporated into every cell, causing not every cell to become ampicillin resistant. The transformation efficiency is calculated in order to tell us the number of transformants per microgram of DNA. Our transformation efficiency was in the range we expected, with 2.17*10^3 being a sensible number of transformants in each microgram of pGLO.


Conclusion: The conclusion we got was exactly what we were expecting it to be. The +pGLO LB/amp/ara plate had dots of bacteria basically covering the whole surface. Once we shined the ultraviolet light on it, it became green, so we knew we did it right. With all the combinations of substances in this one, is what made it glow. The +pGLO LB/amp had dots of bacteria as well, but didnt glow like the other one. The -pGLO LB/amp had no growth on it at all. And -pGLO LB was almost covered completely with bacteria. 




Friday, December 20, 2013

Cellular Communication Lab

Purpose: The purpose of this lab was to determine the effects of varied amounts of time on each type of yeast cell. The different types of yeast cells were A type, alpha type, and mixed. The independent variable was the amount of time the cells were left, and the dependent variable was the number of cells left at the end.

Introduction: Yeast cells use cellular communication to mate and form shmoos. Cellular communication is a method that cells use to talk to each other and mate. Specifically in yeast cells, the A type cells send out a signaling molecule specific to the receptor of the alpha type, and this forms a diploid zygote, which then turns into a diploid budding zygote and forms an ascus. An ascus is a mixture of alpha and A type cells. These types of cells  are only seen when a and alpha type yeast cells are mixed together. When on their own, we only see single cells and budding cells.
Mixed a and alpha cells are shown here. This we can see because there are many more cells viewable through the microscopes.

This shows the same mixture, yet fewer yeast cells can be seen. 

This is alpha type at 48 hours.

Methods:
To begin the experiment, we got culture tubes and labeled them alpha-type, a-type, and mixed. We had 4 mL of sterile water in our culture tube, where then we put a small amount of yeast into each tube and mixed it around thoroughly to make sure all then yeast got into the water.  We were not allowed to use the same pick to get the yeast out for the alpha and a type, but for the mixed we could use either one. Then we went back to our lab table and used a piper to put 5 drops of yeast onto the designated slides. Once again, we couldn't use the same piper for alpha and a type. We put the coverslip over the drops of yeast that was in the slide next. We had to record the yeast at 0 time, 30 minutes, 24 hours and then 48 hours. Once we took out our microscopes and had them all set up, we put one of the slides and focused the yeast using the 10x lens, then once we saw the yeast cells, we changed the lens to objective 40x. Next, we took pictures through the microscope lens so then its would be easier to count all of the cells. We repeated these steps 3 other times per each type of yeast cell. 

Data:
First, we have the results for the mixture of a and alpha yeast cells. 
Then, we have the data for each type individually.


Graphs:

Below is the graph for alpha type yeast over time. The two lines depict single haploid cells and budding haploid cells, as per the key above. 
Then, we have the graph for a-type yeast. 
Below is the key for the mixed yeast culture. 



Discussion:
 We had two types of yeast cells, a and alpha, that were pre-labelled for our lab. If we did not know which was which, but we had one known sample, we could determine each type. For example, say we had a sample of a type yeast cells. When we combine it with other a type cells, we will only see single and budding cells. But if it is combined with alpha cells, shmoos would be visible under the microscope. This is because yeast cells have g-coupled protein receptors. In yeast, these receptors can receive signaling molecules from the opposite type of cell. Once this happens, kinases are activated that stimulate the growth of the cytoskeleton in the direction of the signal. This is where shmoos is created. For our results, there were some obvious patterns. First of all, the graphs of single and budding haploid cells for the a and alpha type cells were always inversely related. As one type's concentration went up, the other decreased. For a-type, the last reading could be a mistake. The microscope itself maybe have been too dirty or malfunctioning, but no other cells were visible as the screen was very dark. Looking at the mixed culture, we can see that the concentration of single haploid cells decreases sharply, which was expected. The number of budding zygotes and asci cells increased over time, which makes sense because a and alpha cells will be communicating with each other in the mixture. If they are mixed, then their proximity allows them to receive signals and mate. The only surprising result was that of the shmoos, which we thought would increase over time rather than stay relatively flat. 




Conclusion:
The conclusion that we had was that obviously the mixed had a lot more yeast cells than the alpha and a type since it was a mixture of both of them. The other observation that we made was that the more time that elapsed, the less percentage of each cell in the mixed, except for the schmoos. Which could be what was supposed to happen or it could be a calculation error in some way. Also I believe the single haploid was eventually supposed to be less than the budding haploid, because they would all turn into those toward the end. That ended up happening in the alpha type and the a type, but the mixed culture seemed to have some differing results.






Monday, December 9, 2013

Plant Pigments and Photosynthesis Lab

Purpose: The purpose of this lab was to measure the different rates of photosynthesis in isolated chloroplasts using DPIP. We used a dye-reduction technique, which shows that light and chloroplasts are necessary for the light reactions to occur. The independent variable is boiled versus unboiled chloroplasts and the amount of time spent in the light. The dependent variable is  the percent transmittance. The control group is the group with no DPIP and the group with no chloroplasts. We were trying to see if the amount of time that each group of chloroplasts was left in the light would effect the percent transmittance.
For the chromatography portion, we wanted to see the distance that pigments in spinach leaves would travel with a solvent. 

Introduction: DPIP was the compound we used in place of the electron acceptor NADPH in photosynthesis. The dye-reduction technique was what showed us how many reactions took place in each group. Each time the DPIP accepted electrons, or was reduced, DPIP changed from blue to colorless.  This means that the more color was lost, the more reactions were taking place. The percent transmittance shows how much light was absorbed and how much was allowed to pass through. 

Methods: To set up the lab we had a flood light, in front of a heat sink, then eventually our five cuvettes would be lined up behind the heat sink. We first got our Labquest 2 set up and attached our colorimeter to it. We had five cuvettes that were all filled differently with our solutions. The 1st cuvette was the blank control and we were using that to collaborate our colorimeter. That had 1 mL of phosphate buffer, 4mL of distilled water, and 3 drops of unboiled chloroplast. The next was one that had unboiled chloroplasts dark which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of unboiled chloroplasts. Cuvette 2 was then covered in foil so that no light could get through to the solution. The 3rd cuvette was unboiled chloroplasts light, which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of unboiled chloroplasts. Cuvette 4 boiled chloroplasts light which had 1 mL of phosphate buffer, 3 mL of distilled water, 1 mL of DPIP, and 3 drops of boiled chloroplasts. The 5th cuvette was the no chlorplasts light, which was our negative control group. This had 1 mL of phosphate buffer, 3mL + 3 drops of distilled water and 1 mL of DPIP. We set the colorimeter to 1 by using the 1st. We weren't able to put the chlorplasts in until we were about to start the procedures. So we put all the chlorplasts in th colorimeter one at a time and at 0 min we measured the transmittance for all 5 cuvettes. Then we put all five in front of the heat sink where the light was shining through. Then at 5, 10, and 15 minutes, we took the cuvettes away from the light to measure the transmittance again and then put it back after each test. 
Test tubes receiving DPIP


Colorimiter being calibrated before the cuvettes were tested for time 0
























The cuvettes and flood lamp




















































Below are pictures shown from the Chromatography Lab. Here, we used cells from spinach leaves on paper, with the end dipped in a solvent, to assess the pigment distribution.




Data:
For the chromatography lab, we have measurements of each band seen in the picture above. 

 This means that the Rf factor for the first band is 0.14, the second band is 0.27, the third is .35, the fourth is .51, and the final is the solvent front.
Then, we have our measurements from the Photosynthesis lab. In the first chart, we have our measurements from the first round of trials. Then, we have measurements from the second round of trials. The significant differences In results will be explained. 



Graphs and Charts:

Using this key, two graphs are shown. The first graph is from the first round of results, and we know these are inaccurate since transmittance can not be greater than 100%. The second graph depicts results from the second round of trials. 



Discussion:
In our first run through of the photosynthesis lab, we immediately knew something was wrong. In our first measurements at time 0, we had transmittance readings over 100%, which is not possible. For this reason, we increased the concentration of chloroplasts from our first round of trials. This way, the reactions would not be nearly finished taking place before we even took measurements in the colorimeter. After making this change, we saw an improvement in our results. Test tube 2, with unboiled dark chloroplasts, remained fairly constant. This makes sense due to the necessity of light for photosynthesis to take place and DPIP to be used. The transmittance of test tube 3, unboiled light chloroplasts, increased over time. This also makes sense due to the fact that the chloroplasts are functioning, and provided light to fuel their reactions. Test tube 4, boiled light chloroplasts, increased initially and then decreased. This is interesting because boiled chloroplasts become denatured, and therefore do not react. The graph should be relatively flat, and more trials could be taken to determine if a mistake was made. Finally, test tube 5 had no chloroplasts, and therefore no reactions taking place, which resulted in little or no change in the amount of transmittance. Excluding test tube 4, these results turned out the way we had hypothesized. 

In the chromatography lab, we used the below formula to determine the Rf factor for each pigment. The results come out different for each pigment because they are not equally soluble in the solvent. Beta carotene traveled the furthest because it was the most soluble, forming no hydrogen bonds. Xanthophyll, on the other hand, is less soluble and forms hydrogen bonds with cellulose, causing it to be found further from the solvent front. We did not have extensive prior knowledge about pigments such as these, but the information provided supported our results.



Conclusion: Cuvette 1 stayed similar in it's transmittance rates because it didn't have DPIP, and acted as the NADP+, which is the electron carrier in photosynthesis. Cuvette 2 didn't have any light going through to it so therefore there were no electrons being produced,so the color didn't change. Then Cuvette 4, which had unboiled chlorplasts which didn't cause much electron production or color change since some proteins had probably been denatured through the process of boiling them. Then, Cuvette 5 was our negative control that didn't have either type of chlorplast, so therefore it didn't have anything to react with and it basically had very similar transmittance each time like Cuvette 2 had.